Abstract
Many living organisms remove wide range of DNA lesions from their genomes by the nucleotide excision repair system. The uvrB gene, which plays an essential role in the prokaryotic excision repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence was determined, and the deduced amino acid sequence showed it possessed a helicase motif, including a nucleotide-binding consensus sequence (Walker's A-type motif), which was also conserved in other UvrB proteins. The prokaryotic UvrB proteins and eukaryotic DNA repair helicases (Rad3 and XP-D) were classified into different groups by molecular phylogenetic analysis. The T. thermophilus uvrB gene product was overproduced in Escherichia coli and purified to apparent homogeneity. The purified T. thermophilus UvrB protein was stable up to 80 degrees C at neutral pH. T. thermophilus UvrB protein showed ATPase activity at its physiological temperature, whereas the E. coli UvrB protein alone has not been shown to exhibit detectable ATPase activity. The values of K(m) and k(cat) for the ATPase activity were 4.2 mM and 0.32 s-1 without DNA, and 4.0 mM and 0.46 s-1 with single-stranded DNA, respectively. This suggests that T. thermophilus UvrB protein could interact with single-stranded DNA in the absence of UvrA protein.
Highlights
All living organisms have DNA repair systems to counteract the many forms of DNA damage due to sunlight, chemical agents, or ionizing radiation [1]
Hyper-thermophilic bacteria can grow at temperatures over 100 °C, T. thermophilus is the most thermophilic bacterium whose gene manipulating system has been established among thermophilic bacteria
The purity of the final fraction of T. thermophilus UvrB protein was over 99%, based on the densitometry of SDS-polyacrylamide gel stained with Coomassie Brilliant Blue (CBB) (Fig. 4B, lane 5) or silver-staining, N-terminal amino acid sequencing analysis and the size exclusion chromatography (Fig. 8)
Summary
Enzymes and Chemicals—The enzymes and reagents were purchased as follows: DNA modification enzymes, including restriction endonucleases, from Takara Shuzo, Nippon Gene, Toyobo, and New England Biolabs; Taq DNA polymerase from Perkin Elmer; isopropyl1-thio--D-galactopyranoside (IPTG) from Wako Pure Chemical; DEAEcellulose DE52 from Whatman Biochemicals; Phenyl-Toyopearl 650 M from Tosoh; hydroxylapatite from Nacalai Tesque; [␣-32P]ATP from ICN; plastic-backed polyethyleneimine cellulose sheets (MN-Polygram CEL200PEI/UV) from Machery and Nagel; poly(dC) from Pharmacia Biotech Inc. The fractions containing the protein were pooled and dialyzed against buffer III (1 mM EDTA, 10 mM -mercaptoethanol, 10% (v/v) glycerol) containing 10 mM potassium phosphate (pH 6.8), and loaded onto a column of hydroxylapatite (bed volume 80 ml) equilibrated with this buffer. The fractions were analyzed as described above, and UvrB protein was eluted at 120 mM potassium phosphate These fractions were pooled and dialyzed against buffer II containing 100 mM KCl, and stored at 4 °C. The purity of the final fraction of T. thermophilus UvrB protein was over 99%, based on the densitometry of SDS-polyacrylamide gel stained with Coomassie Brilliant Blue (CBB) (Fig. 4B, lane 5) or silver-staining, N-terminal amino acid sequencing analysis and the size exclusion chromatography (Fig. 8). The sheets were placed in contact with imaging plates for 20 min, and the radioactive counts due to ATP and ADP were determined using a BAS2000 image analyzer (Fuji photo film)
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